Project Summary An intriguing phenomenon is observed in many neuroinflammation-based conditions, where neuroinflammatory responses and neurophysiological changes occur distant to a focal brain lesion. This contributes to the therapeutic challenges in managing neuroinflammatory diseases. Due to large diffusion distances, soluble factor-based signaling is unlikely to cause distal neuroinflammatory responses, while focal neuroinflammation is too specific to be explained by vascular transport of soluble factors. Our central hypothesis is that intra-axonal signaling and electrophysiological signals (e.g., excitotoxicity) contribute to the transmission of neuroinflammatory triggers from the site of insult to distal regions. Distinguishing the relative contributions of these complex factors in vivo, where many confounding signals exist, is extremely difficult if not impossible. However, conventional in vitro tissue culture models are also inadequate for addressing this problem because they do not recapitulate the spatial relationship of this phenomenon. In order to address this need, we will employ a microfluidic in vitro model that consist of two physically distinct culture chambers (e.g., source and target, corresponding to immediate and distal anatomical regions) interconnected by microchannels. This platform will allow for organotypic brain slice culture and the two chambers will be electrically connected by axonal projections routed through the microchannels, while the chambers will remain chemically separated by the high fluidic resistance of the channels and differential hydrostatic pressure at each chamber. Each transparent chamber and the microchannels will allow for monitoring histological and biochemical changes and each will contain multifunctional multiple electrode arrays for monitoring electrophysiological activity, allowing us to assess the relative contribution of intra-axonal signaling and electrophysiological signal to the propagation of neuroinflammation between discrete brain regions. Collectively, the pilot study is expected to (i) validate the propagation of neuroinflammation observed in vivo and (ii) establish the foundation for future mechanistic studies of neuroinflammation and its intra-axonal transmission with unprecedented control and detail.